Formation of ethyl acetate by Kluyveromyces marxianus on whey during aerobic batch cultivation at specific trace element limitation

Abstract

Kluyveromyces marxianus is able to transform lactose into ethyl acetate as a bulk product which offers a chance for an economical reuse of whey-borne sugar. Ethyl acetate is highly volatile and allows its process-integrated recovery by stripping from the aerated bioreactor. Extensive formation of ethyl acetate by K. marxianus DSM 5422 required restriction of yeast growth by a lack of trace elements. Several aerobic batch processes were done in a 1-L stirred reactor using whey-borne culture medium supplemented with an individual trace element solution excluding Mn, Mo, Fe, Cu, or Zn for identifying the trace element(s) crucial for the observed ester synthesis. Only a lack of Fe, Cu, or Zn restricted yeast growth while exclusion of Mn and Mo did not exhibit any effect due to a higher amount of the latter in the used whey. Limitation of growth by Fe or Cu caused significant production of ethyl acetate while limitation by Zn resulted in formation of ethanol. A lack of Fe or Cu obviously makes the respiratory chain inefficient resulting in an increased mitochondrial NADH level followed by a reduced metabolic flux of acetyl-SCoA into the citrate cycle. Synthesis of ethyl acetate from acetyl-SCoA and ethanol by alcoholysis is thus interpreted as an overflow metabolism.

Efficient growth of Kluyveromyces marxianus biomass used as a biocatalyst in the sustainable production of ethyl acetate

“…Precipitated minerals were allowed to settle overnight before the upper phase was withdrawn for cultivation experiments. Nonsupplemented DW medium should not be confused with DW basic medium (as used in [11][12][13][14]16]) which contains 10 g/L (NH 4 ) 2 SO 4 .…”

“…One reference experiment was done with DW basic medium (DW medium with 10 g/L (NH 4 ) 2 SO 4 ) and described in detail in [11] while all other presented bioreactor processes were based on non-supplemented DW medium. The sterile 1-L stirred bioreactor with 0.6 mL Antifoam A (Fluka, Sigma Aldrich, St. Louis, USA) was filled with 0.6 L DW medium and 1.2 mL trace-element solution (the latter described in [10]).…”

“…Various yeast species can synthesize ethyl acetate (reviewed by Löser et al [9]), but only Pichia anomala, Candida utilis, and Kluyveromyces marxianus produce this ester in larger amounts. K. marxianus is the most promising candidate for large-scale ester production since this dairy yeast with GRAS status grows quickly, converts sugar directly into ethyl acetate without ethanol as an essential intermediate, and produces the ester with a high rate and yield [9][10][11][12][13][14][15][16][17]. The ester synthesis in K. marxianus is easy to control by the level of iron [11,13,[16][17][18].…”

“…K. marxianus is the most promising candidate for large-scale ester production since this dairy yeast with GRAS status grows quickly, converts sugar directly into ethyl acetate without ethanol as an essential intermediate, and produces the ester with a high rate and yield [9][10][11][12][13][14][15][16][17]. The ester synthesis in K. marxianus is easy to control by the level of iron [11,13,[16][17][18]. K. marxianus exhibits a distinct thermal tolerance which allows cultivation at an elevated temperature [14,19,20] which in turn accelerates the ester stripping and advances its process-integrated recovery.…”

“…Whey was often supplemented with ammonium as a source of nitrogen to stimulate growth of K. marxianus [10][11][12][13][14]16,27,29,[31][32][33][34][35][36][37]. Added ammonium increased the yield of biomass [27,32,35] or was without significant effect [29,31,36].…”